160 million women develop Breast Cancer (BC) annually with subsequent 500,000 deaths, and therefore it is the second most common cause of cancer related deaths in women. Improved treatments together with earlier diagnosis made the disease more manageable, however 30 –40% of patients will eventually develop metastasis. Metastasis prognosis remains poor, and new therapies effective against advanced and disseminated breast cancer are needed. Bone is the third most common site for metastatic cancers, after lung and liver. Understanding the molecular pathways promoting bone metastasis may offer new therapeutic targets. Silencing of interferon-α (IFNα) signalling was recently reported as one of the breast cancer bone metastasis hallmarks. Reversing this, may inhibit bone metastasis formation and progression. IFNα is a potent immune stimulator and anti-tumoural molecule, however, its clinical use is limited by the systemic toxicity associated with its therapeutic doses. Therefore, novel therapeutic strategies are essential for the tumour target delivery of IFNα. Our lab together with Prof Luigi Naldini (TIGET, Milan, Italy) have developed a novel platform for the target delivery of bio-therapeutic molecules to tumour tissues or metastatic organs. This strategy is based on autologous transplantation of gene-modified haematopoietic stem/progenitor cells (HS/PC) expressing the IFNα gene under the control of a cell-specific promoter: the Tie2 promoter. Transplanted HS/PCs then differentiate into all haematopoietic lineages including a subset of tumour-infiltrating monocytes (Tie2-expressing monocytes, TEMs), which specifically upregulate Tie2 after homing to tumour tissues. They previously demonstrated its ability to inhibit primary tumours and lung metastasis in a mouse model of Luminal B breast cancer. They also developed the human delivery platform and showed inhibition of human primary TNBC in a humanised preclinical model. Of note, tumour targeted delivery of IFNα had a positive toxicity profile unlike its systemic administration. Here, we show that this IFNα-delivery strategy was also able to inhibit TNBC lung and bone metastasis in an immunocompetent preclinical murine model and in a humanised mouse model. Moreover, we tested its ability to improve the outcome of immune checkpoint modulators, or cancer vaccination. As immune checkpoint modulators, we used a new combination currently under investigation in our laboratory that involves anti-PD-1 and anti-41BB antibodies. As a cancer vaccination strategy, we used a novel platform developed in our laboratory for the in vivo delivery of relevant antigen formulations to Clec9A+ dendritic cells and based on tailorable oil-in-water nanoemulsion (Clec9A-TNE). Our results show that this IFNα delivery strategy was able to improve the outcome of cancer vaccination but did not synergies with anti-PD1 and 41-BB. Finally, thinking about a possible clinical translation, we explored the use of adoptively transferred Tie2-IFNα mature monocytes for the tumour targeted delivery of IFNα in combination with low doses of chemotherapy (Doxorubicin) and showed a significant improvement in the survival of mice treated with this novel combination therapy. Overall, our data support the clinical translation of our gene- and cell-based therapy for the tumour targeted delivery of IFNα, which has a good safety profile and can enhance therapeutic efficacy when given in combination with best-matching therapies.

The tumor immunosuppressive microenvironment is the main challenge to cancer immunotherapy efficacy. By transducing HSCs with a Tie2-interferon-alpha (IFNα) transgene, we converted a tumor homing monocyte subpopulation characterized by the expression of the Tie2 receptor (TIE2-expressing macrophages: TEMs) into an effective tumor-targeted delivery vehicle for this potent immune-stimulatory molecule. This approach intensely repressed both primary and lung metastatic breast cancers in mouse and human hematochimeric models. Because Type I IFN silencing has a key role in maintaining breast cancer bone metastasis, we hypothesized that our IFNα delivery approach could efficiently inhibit this lethal form of breast cancer. Toward this goal we established new tumor cell lines (4T1.2sLuc) that steadily secrete Gaussia Luciferase (sGluc) in the blood and urine to facilitate the longitudinal measurement of total metastatic burden over time. The sGluc was detected in the blood of tumor bearing mice for the first few days, but quickly dropped down despite a normal tumor growth. Further investigation is required to determine the mechanism responsible for the observed decrease in sGluc signal. Moreover, with the final goal of investigating whether adoptive transfer of genetically engineered monocytes could be considered as an alternative to HSCs transplantation for the delivery of IFNα, we are developing efficient isolation protocols for adult monocytes. We identified the spleen of tumor bearing mice as a rich source of donor monocytes. However, further optimization of the isolation protocol is required. Finally, preliminary data suggest that adoptively transferred mature monocytes are able to home to primary mammary tumors and develop into macrophages.

The study was carried out in buffalo to give a comparative diagnostic imaging analysis (radiography, ultrasonography, computed tomography and magnetic resonance imaging) regarding to stifle sectional anatomy and corresponding anatomical dissection. The key target was to provide anatomical explanation for stifle intra-articular anatomical disorders by utilising different visualization aids. This study will help veterinary surgeons to determine the appropriate surgical intervention basing on the communication between the femoropatellar, medial femorotibial, and lateral femorotibial synovial sac compartments.